Fig. 5: CD4 clusters represent the receptor accumulation at the tips of membrane protrusions.
From: Approach to map nanotopography of cell surface receptors
a Representative three-dimensional dTRABI image of CD4 WT at the surface of Jurkat cell immobilized on a glycine-coated coverslip. b The axial distribution of CD4 WT localizations of the 4 μm² region of interest (ROI) of the cell in a. c Representative 3D dTRABI image of non-palmitoylatable CD4 CS1 mutant at the surface of Jurkat cell immobilized on a glycine-coated coverslip. d The axial distribution of CD4 CS1 localizations of the 4 μm² ROI of the cell in c. e–g Quantitative analysis of the receptor axial (z-axis) distribution on the surface of T cells. Receptors were analyzed by using a bi-Gaussian fit to the axial distribution of localizations for each ROI as in b, d and i (see Supplementary Fig. S11 for more examples and Supplementary Data for the details) and the FWHM range of the two Gaussian functions represent the z-distribution width (zw) as depicted in e. Black circles in e represent the axial distribution of receptor localizations for a selected ROI, black line the bi-Gaussian fit, which is the sum of two Gaussians as depicted in red and blue, dashed lines in gray depict zw and lines in light gray depict mean values of the Gaussian distributions. The graphs in f and g represent histograms of zw obtained from 21 CD4 WT cells with 846 ROIs (black), 18 CD4 CS1 cells with 1044 ROIs (green) and 13 CD45 WT cells with 305 ROIs (magenta). The histograms in f and g show relative and absolute occurrence, respectively. Data points in b, d, f and i were spline interpolated to guide the eye. h Representative 3D dTRABI image of CD45 at the surface of Jurkat cell immobilized on a glycine-coated coverslip. Color-bars in the upper right corner of a, c and h indicate the axial position of the localizations in the image. i The axial distribution of CD45 localizations of the 4 μm² ROI of the cell in h. CD4 variants and CD45 were stained with directly labeled primary antibody as in Figs. 3 and 4, respectively. Scale bars in a and c and h, 5 μm.
